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Researchers at the Dana-Farber Cancer Institute have identified a glutamine metabolism pathway that is
activated by oncogenic K-Ras
in pancreatic cancers.1 Moreover, the team found three different
enzyme targets within the pathway that could be blocked to specifically inhibit
proliferation of the malignant cells.

Cancer cells have
higher metabolic demands than normal cells due to their high levels of growth
and proliferation. This increased rate of glycolysis, dubbed the Warburg
effect, helps sustain tumor growth.

Recent studies have shown that some oncoproteins,
including c-Myc
(MYC)
and K-Ras, which are known to activate genes involved in cancer cell
proliferation, also act as drivers for cancer metabolism.

In a 2012 paper published in Cell, a Dana-Farber
team led by Alec Kimmelman identified two K-Ras-activated glucose metabolism
pathways in pancreatic cancer cells. Inhibiting the pathways helped stop cancer
cell proliferation.2

The K-Ras oncogene is expressed in various cancer
types, including about 90% of pancreatic cancers. It is associated with poor
prognosis and causes resistance to cancer drugs including epidermal
growth factor receptor (EGFR)
inhibitors. Attempts to target the oncogene directly have not been successful
due to the complex biology and interactions of the enzyme's mutant form.

Building on their previous work, Kimmelman and colleagues
found that K-Ras activated an anabolic glutamine metabolic pathway specifically
in the cancer cells that not only generated energy and building blocks for
protein synthesis but also regulated redox homeostasis to allow cancer cell
growth.

Healthy cells employ a glutamine metabolic pathway that
converts glutamine-derived glutamate to a-ketoglutarate
(a-KG) using the
enzyme glutamate dehydrogenase 1 (GLUD1).
a-KG is then used to
fuel the tricarboxylic acid (TCA)
cycle to create energy. In healthy cells, glutamine also can be processed by transaminases
in an anabolic pathway that results in both a-KG
and the synthesis of nonessential amino acids (NEAAs).

The researchers wanted to probe whether cancer cells were
dependent on either of these pathways (see"Glutamine metabolism").

In pancreatic ductal adenocarcinoma (PDAC) cells,
glutamine deprivation or inhibition of glutaminase
(GLS),
the enzyme that converts glutamine to glutamate early in metabolic processing,
suppressed cell growth. Addition of NEAAs plus a-KG, but not a-KG
alone, restored cell growth in the glutamine-deprived cells.

These findings suggest that the cancer cells depend on
the alternative transaminase-dependent glutamine metabolism pathway for
proliferation.

For confirmation, the team showed that a nonspecific
transaminase inhibitor or a specific inhibitor of the aspartate transaminase glutamic-oxaloacetic
transaminase 1 (GOT1) decreased cell
growth compared with vehicle. Inhibition of GLUD1 or other transaminases did
not affect cell growth.

The full picture shows that the pathway maintains low
levels of oxidative stress and reactive oxygen species
(ROS)
within tumors. Indeed, knockdown of pathway enzymes increased ROS levels and
inhibited proliferation, whereas treatment with an antioxidant restored cell
proliferation.

In mice with human PDAC xenografts, expression of small
hairpin RNAs targeting GOT1, MDH1 or ME1 suppressed tumor growth, whereas
control shRNA or shRNA targeting GLUD1 did not. In healthy human pancreatic
ductal cells or diploid fibroblasts, inhibition of the enzymes did not alter
cell growth.

Finally, the team drew a connection between oncogenic K-Ras
expression and the anabolic glutamine metabolism pathway in PDAC. K-Ras
knockdown in the cells decreased levels of GOT1 and increased levels of GLUD1,
suggesting that the oncogene upregulates enzymes involved in the alternative
pathway.

Safety and specificity

"Based on our
work, GOT1, MDH1 and ME1 are all potential therapeutic targets," Kimmelman
told SciBX. The team now plans to develop inhibitors of the new targets.

Paul Bingham, VP of research at Cornerstone Pharmaceuticals Inc.,
said, "The most important thing to do next is to find out if you can
attack the malic enzymes and transaminases safely. They have identified really
clear targets, but they are still a long way from showing that you can attack
these enzymes in an intact animal and not cause unwanted toxicities."

He added that the three new enzyme targets upregulated in
tumors are wild-type and thus would be present in some quantities in healthy
cells.

Cornerstone's CPI-613,
an analog of a-lipoic acid
that targets pyruvate
dehydrogenase (PDH)
and a-KG dehydrogenase,
is in
Phase I/II testing with gemcitabine to treat pancreatic cancer.
Bingham said the analog targets multiple activities of the cancer mitochondrial
metabolic pathway including redox metabolism.

Neil Jones, senior principal scientist at Cancer Research UK's
Cancer Research Technology Ltd.
commercial arm, cautioned that "historically, perturbation of the
glutamine pathway has caused some concerns in terms of potential toxicity, especially
in the brain, so it would be important to assess these liabilities as early as
possible in development of potential therapies. The fact that normal cell line
models were not affected by glutamine pathway modulations goes some way toward
addressing this."

According to Susan Critchlow, associate director of
innovative medicines and oncology at AstraZeneca plc,
"Future studies could evaluate whether small molecule inhibitors of the
glutamine metabolism pathway inhibit the growth of established xenograft models
and [could] confirm the expected metabolomics profile is desired."

She noted that the validation studies in the paper only
used genetic knockdown approaches in small tumors.

AstraZeneca and Cancer Research Technology are
identifying cancer metabolism targets and developing therapeutics under a
three-year deal. The partners recently extended the deal for two more years
through the beginning of 2015.

Critchlow thinks inhibitors of the enzymes identified in
the paper could combine well with standard of care for pancreatic cancer, which
involves chemotherapy and radiation. The reason, she said, is that enzyme
inhibition disrupts the ability of cancer cells to cope with oxidative stress,
and standard of care increases intracellular oxidative stress.

She added, "GOT1 shRNA-mediated knockdown
gives rise to a cytostatic effect in vivo, suggesting that combination
with standard of care may be required to induce tumor regression."

Kimmelman agreed. "Our results suggest that since
this pathway is critical for redox balance, there could be synergy with
available therapies that generate reactive oxygen species," he said.

Beyond the pancreas

The link between K-Ras
and the glutamine metabolism pathway could mean that the three new targets are
at work in multiple tumor types.

Jones agreed. "Defining in more detail the potential
patient populations and ascertaining the strengths of the K-Ras lineage against
these targets and whether they would help other K-Ras-driven tumor types would
also help position these targets," he said.

Bingham was less sanguine about the broad applicability
of the findings.

"The metabolic implications of oncogenic K-Ras
are different in different cell lines. K-Ras may not be activating this
particular pathway in other tumor types," he said. "Even if this
strategy proves safe, it might only work for this subset of pancreatic tumors.
However, this is a nasty group of tumors, so it would be no small
accomplishment."

Kimmelman told SciBX that his team has not tested
other cancer types. "However, it is possible that since oncogenic K-Ras
is responsible for regulating expression of key enzymes in this pathway, that
other tumors that have K-Ras mutations may also rely on it for growth,"
he said. "This will need to be studied."

He said Dana-Farber has filed a patent application
covering the work. The IP is available for licensing.

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